Method of checking the performance of a flow cytometer instrument and apparatus for executing said method as well as a standard kit therefore

ABSTRACT

A method for correcting the settings of a flow cytometer, designed for fast sample handling and counting, allowing about 500 samples per hour to be counted. The counting is based on the provision of data representing a PHA diagram (Pulse Height Analysis) of registered pulses, each indicating a passed cell or particle. To check the settings the user measures a standard sample of uniform microbeads  161  on the flow cytometer, and inserts information on a disk  162  in a computer arranged to process the measured data and to calculate: a plurality of particle counts on the same sample, a mean count, a standard deviation s and/or Coefficient of Variation CV, a signal mean value SM, a signal width (width of the bell-curve in the PHA-diagram). The parameters are compared to pre-set limits ( 165, 166, 167, 168 ) and the PHAS curve is compared to an ideal curve PHA 0.  A user help program for adjusting the flow cytometer is arranged to display typical symptoms on a computer screen, to indicate the possible defects and to recommend actions to remedy the problems, based on information in a library stored in the computer. Thereby a visit by a service engineer can often be avoided.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/DK98/00389 which has an Internationalfiling date of Sep. 14, 1998, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention concerns a new method of checking the performanceof a flow cytometer, the flow cytometer comprising means deliveringelectrical pulse signals indicating the presence of particles, such assomatic cells or bacteria. The invention is specifically dedicated tomilk testing and more specifically to determination of the number ofsomatic cells in milk. Further according to the present invention themethod allows for inspection and correction for any agglomeration ofparticles in a standard sample used for checking the performance of theflow cytometer. Further the invention concerns an apparatus comprisingdata processing equipment arranged to execute the above method, as wellas a stannard kit for application in the apparatus.

BACKGROUND ART

Flow cytometers are instruments used for analysis of particles suspendedin a fluid, e.g. biological cells in body fluids, such as somatic cellsin milk. Briefly, a flow cytometer comprises a liquid flow system and anoptical system intersecting each other in a flow cuvette, as well as anelectronic system, detecting fluorescence or scatter originating fromparticles passing through the cuvette. Preferably, the fluid sampleflows in a single, thin string inside the cuvette thereby allowing anyparticles present to be counted one by one. Further, in a preferredembodiment the thin string is surrounded by a sheath fluid. Thanks tothe sheath fluid, casual impurities, present in the fluid and having asize larger than the particles or cells to be counted and accordinglylarger than the size of the string of fluid sample, will not causeclogging. They might have done so if flowing in a liquid channel asnarrow as the thin inner string.

The method according to the invention is specifically developed for flowcytometers applied for fast determination of the number of cells in amilk sample or a milk product and more specifically for the fluorescenttype of flow cytometers, in which the cells or particles are all stainedby a fluorescent dye, which reacts fluorescent when exposed toillumination. A fluorescence signal of adequate size, i.e. above noise,is considered to indicate the passage of a particle or cell. In thepreferred embodiment of the flow cytometer, the presently preferredagent or dye is Ethidium Bromide. The present type of flow cytometer hasonly one channel, i.e. it is optimised to detect only one kind offluorescence or scatter. However, flow cytometers may have a pluralityof channels, each dedicated to a specific dye.

The somatic cell count is considered a measure of the milk quality (highquality milk has a low cell count). Accordingly the cell count can beapplied by the dairy when setting the price according to which thefarmer is paid for the delivered milk. In order to ensure correctpayment to the farmer and to reveal any milk of too low quality, aproper functioning of the flow cytometer is crucial.

In order to obtain accurate and reproducible results flow cytometersmust be aligned and calibrated. The light ray from the illuminationsource must hit the stream of particles and the detector optics mustfocus on the particle stream when the illuminated particles exhibitfluorescence. Also the gain and characteristics of the electroniccircuits processing the detected signals and the applied mathematicsmust produce a number indicating the true number of particles in thestream with a sufficient accuracy and reproducibility.

Possible Reasons for Poor Performance

Any defects or maladjustment of flow rates (or clogging) in the flowsystem and/or defects or misalignment of the optical system may causethe count to either decrease or increase compared to the true value.

Prior Art

U.S. Pat. No. 5,093,234 (Schwartz) discloses a method of aligning,compensating and calibrating a flow cytometer for analysis of samplesand a microbead standard kit therefore. The method can be applied tomulti-channel flow cytometers. The method includes running testmeasurements on standard kits; adjusting fluorescence channel PMTvoltages and gain to position resulting dot plot or histogram near theorigin of the axis of each of the fluorescence channels; and settingboundary levels in each channel to indicate fluorescence intensity. Thestandard kit comprises a blank and/or an auto-fluorescent microbeadpopulation and at least two series of calibrated microbead populationslabelled with fluorescent dye(s). U.S. Pat. No. 5,084,394 (Vogt et al)discloses a similar method for corrective calibration of a flowcytometry using a mixture of fluorescent microbeads and cells. Thesemethods provide for an advanced, sophisticated and qualitative analysisof single cells such as lymphocytes in blood.

The present method concerns a “performance check”, i.e. a monitoring offunctional performance, of a single channel flow cytometer for countingsomatic cells in milk. The instrument is specifically designed for fastsample handling and counting, allowing about 500 samples pr hour to becounted. This kind of flow cytometer counts the cells based uponmeasuring only one parameter, such as green or red fluorescence. Theinstrument is not intended for qualitative studies of the cells. Theperformance check method is based on the use of a standard and/orcalibration fluid comprising only one type of particles or microbeadswhich are unstained until they enter the process according to thepresent invention. Thereby the standard fluid samples are as simple aspossible. The standard samples are very stable and adequate for a longterm shelf life, i.e. a great number of standard samples may be storedby the user for months or years for future use, such as a regularperformance check every morning or when ever necessary.

Besides a thorough check of the operation of the flow cytometer thestandard fluid could also be applied for a calibration of the flowcytometer.

SUMMARY OF THE INVENTION

The present invention provides a method of checking the performance of aflow cytometer instrument, in which instrument the number of particlesor cells in a fluid flow are counted by providing data representing aPHA diagram (Pulse Height Analysis), of registered pulses. The inventionis characterised by—providing a lot of standard samples, including onlyone type of substantially uniform microbeads,—providing datarepresenting an optimal (desired) PHA diagram (PHA0) of the pulsesregistered, when measuring a standard sample from the lot on a referenceinstrument, said data being stored in a memory in the instrument itself,or a memory in data processing means connected to the instrument, or inmeans from which the data may be imported into the instrument or intothe data processing means connected to the instrument,—measuring astandard sample on the instrument to be checked,—providing datarepresenting a PHA diagram (PHAS) for the pulses registered during themeasurement of the standard sample on the instrument to bechecked,—comparing the present PHA diagram (PHAS) to the optimal PHAdiagram (PHA0),—and analysing and/or evaluating said data in order todetermine any poor or faulty operation of the instrument. By this methodthe user can check the instrument regularly, and the user can readily beinformed of any precautions to be taken. Preferably the microbeads inthe lot are unstained until the enter the instrument. The use ofunstained microbeads are specific favourable in that also the stainingprocess in the instrument is controlled when measuring the standardsample.

Preferably at least one of the following parameters are calculated: aparticle count, a plurality of particle counts on the same sample, astandard deviation, s, and/or Coefficient of Variation, CV, based on (atleast two, preferably three) repeated/consecutive measurements on thesame sample and substantially at the same time, a signal mean value, anda signal width, i.e. the width of the bell-curve in the PHA-diagram—thecorresponding data for the standard sample of the standard fluidmeasured on a reference instrument. i.e. the optimum values of saiddata, being provided with the lot—comparing at least one of the aboveparameters for the actual measurement of standard sample to thecorresponding optimal parameters of the standard fluid,—and analysingsaid data to estimate whether the instrument is operating substantiallyoptimally, (i.e. within preferred limits) or is not operatingsubstantially optimally (i.e. outside preferred limits), registering anyoff-limit deviations from optimal operation.

Preferably the registered off-limit deviations are considered assymptoms which are displayed to the user.

Preferably the data processing equipment comprises means for evaluatingthe observed symptoms, proposing possible defects (make a diagnosis),and making recommendations for how to remedy any poor performance,and/or any precautions to be taken. Preferably the means for evaluatingthe symptoms include a library stored in memory means arranged to beaccessed by the data processing means. Preferably, the means forchecking the performance/evaluating the symptoms includes display of alist of consecutive results to the user on a monitor and/or providing aprint out. Preferably, the means for checking the performance/evaluatingthe symptoms includes display of detailed results and data of recentmeasurements of the standard fluid. Preferably the means for checkingthe performance/evaluating the symptoms includes displaying a measuredPHA—diagram on request from the user.

Preferably the means for checking the performance/evaluating thesymptoms includes display of a list of possible situations comprisingdefined symptoms and for each situation present information on possiblereasons and appropriate actions to remedy the defect.

The display of any symptoms and actions to remedy any default operationis advantageous in that the user himself will be able to adjust theinstrument to obtain optimum performance. Thereby the visit by a serviceengineer can be avoided. Thereby the loss of precious measurement timedue to poor performance is avoided.

The apparatus according to the invention is arranged to execute theabove method. The standard kit according to the invention comprises astandard fluid including a plurality of substantially uniform microbeadsand associated means carrying information on the lot, and specificallyon the content of microbeads in the standard fluid.

The method and kit is specifically favourable in that the test samples(standard samples) are handled in the same way as any other sample, andany defect in the instrument, which is liable to influence an ordinarymeasurement result will also be liable to in influence the result of thetest measurement on the standard sample.

Further scope of the applicability of the present invention will becomeapparent from the detailed description given hereinafter. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given hereinbelow and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein:

FIG. 1 shows a schematic and simplified diagram of a flow cytometeraccording to the invention.

FIG. 2 shows a typical PHA diagram.

FIG. 3 shows a display of PHA diagrams according to the inventionindicating e.g. that gain setting may be a little too high,

FIG. 4 shows another display of PHA diagrams according to the inventionindicating e.g. too high flow rate of sheath fluid.

FIG. 5 shows a diagram indicating the variation of counts compared withlot count.

FIG. 6 shows a diagram indicating the signal mean compared with lot.

FIG. 7 shows the standard deviation or Coefficient of Variation, CV,compared with theoretical CV.

FIG. 8 shows a cumulative mean count compared with theoretical values(Count Cumulative).

FIG. 9 shows a screen print displaying three “windows”, 1) a list ofresult, 2) result details, and 3) lot data, as the screen print mayappear unclear all data are listed in tables in the text.

FIG. 10 shows the PHA-diagram for a specific measurement (No 58.2).

FIG. 11 shows a screen print showing the PHA-diagram for a specificmeasurement (No 58.2) and a theoretical, desired PHA-diagram.

FIG. 12 shows a plot of a PHA-diagram for a first specific situation.

FIG. 13 shows a plot of a PHA-diagram for a second specific situation.

FIG. 14 shows a plot of a PHA-diagram for a third specific situation.

FIG. 15 shows a PHA-diagram indicating agglomeration of particles.

FIG. 16 shows a schematic diagram illustrating the method according tothe invention.

The screen prints shown in FIGS. 9 and 11 may appear a little hazy, andfor the sake of clarity all relevant data are presented in tables 1, 2and 3.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The invention will be explained in greater details in the followingnon-limiting example of a preferred embodiment according to theinvention.

The Flow Cytometer

A preferred embodiment of a flow cytometer according to the invention isshown schematically in FIG. 1. The flow cytometer 10 comprises a flowsystem 12 adapted to aspirate a milk sample from a sample cup 14; theflow system is arranged to mix the sample with a fluorescent dye. Theflow system includes a transparent cuvette 16 through which a thinstring of the milk sample is passing surrounded by a sheath fluid. Anoptical system 18 includes a light source 20, optics forwarding a lightbeam, passing through the transparent cuvette 16, hitting the thinstring of the milk sample, further optics collecting any fluorescencelight emitted by the stained cells in the milk sample passing throughthe cuvette 16, a photo detector 24 detecting the fluorescence light,i.e. transferring the fluorescence light into electronic signals, andelectronic signal and data processing equipment 26. Preferably, the dataprocessing equipment includes a personal computer 28. Further the dataprocessing equipment includes a monitor 30 or similar display unit. Theflow cytometer is specifically designed for use in dairies andlaboratories analysing milk supplied by farmers.

The features of the method are illustrated schematically in FIG. 16 andexplained in details in the following: . . .

The Lot

The user of the flow cytometer receives from the flow cytometermanufacturer or other supplier a so-called “lot”, which is a batch ofstandard samples, i.e. a plurality of small containers filled with astandard fluid. The characteristics of the standard fluid have been dulyanalysed by the supplier. The presently preferred standard samplescomprise 30 ml water, additives and a number of plastic beads, about 6μm of size. As the plastic beads tend to settle on the bottom of thesample cup, careful shaking of the presently applied standard samples isessential to obtain a good result.

The standard fluid may also be applied for calibration of the flowcytometer, but such use is not the main item of this patent application.

Information on the characteristics of the standard samples of the lot isdelivered to the user, preferably on storage means, such as a floppydisk or a CD-ROM, also comprising software providing for analysis of thesignals detected and counts obtained by the flow cytometer instrument.The user imports the lot data from the disk to the data processingequipment 26-28 attached to the flow cytometer. Alternatively thestandard kit including the lot may comprise means such as a useridentification and/or password allowing access to storage meanscontaining the information, e.g. information to be accessed by theinternet.

Performance Check and Operation of the Flow Cytometer

Generally, the standard samples are handled and counted in the flowcytometer in the same manner as the ordinary milk samples. Regularly,e.g. about every 2 hours, the user introduces a well shaken standardsample into the flow cytometer. When a portion of the standard samplefluid is aspirated into the flow cytometer it is mixed with afluorescent dye such as Ethidium Bromide. The flow system 12incorporates appropriate mixing and time delay means to ensure that somedye has adhered to each cell or particle to be counted. The stainedcells or particles pass preferably one by one in a string of samplefluid surrounded by a sheath fluid. A light beam from the optic lightsource 20 passes across the string of particles thereby initiating thestained particles to fluorescence. Each flash of fluorescence isdetected by a highly sensitive photo detector 24 connected to the signaland data processing equipment 26-28 wherein the counting isaccomplished.

The Counting and the PHA Diagram

The counting of particles can be analysed by use of the so-calledPHA-diagram (PHA means Pulse Height Analysis). It is a diagram orhistogram showing the number of pulses versus the signal size (height).A typical PHA-diagram is shown in FIG. 2. It appears from the curvesection 32 in the diagram that a great number of low-signal pulses aredetected. Generally, they indicate noise. The bell formed section 34 (inthe middle of the diagram) having a maximum indicates the appearance ofhigher signals, which are interpreted as being caused by the particlesto be counted. The area of said portion indicates the number ofparticles. The PHA-diagram is very useful for indicating the noise andaccordingly enables discrimination of the noise. In a preferredembodiment all signals of a size smaller than an adjustable value D,equal to or close to the minimum M shown in FIG. 2, are considered asnoise. All signals of a size greater than D are considered to representparticles and are accordingly counted.

PHA-diagnostics

According to the present invention the PHA-diagram (FIGS. 2, 3, 4) isextremely useful for diagnosing the performance (operation) of theinstrument. According to the invention the PHA-diagram (or curve)obtained for the standard sample can be displayed to the user on theattached monitor 30 (or display unit) together with a second PHA-diagram(or curve), PHA0. The second diagram or curve indicates the optimal,(desired=best possible) diagram for the measured lot (the second diagramis obtained from the data supplied on the INFO disk forwarded bysupplier together with the lot). If the new PHA-diagram (PHAS=PKA ofStandard Sample) obtained for the standard sample covers or is identical(or almost identical) to the optimal diagram PHA0 the operation of theflow cytometer is good. Any difference can be used for diagnosis asexplained further in the following. Differences exceeding predefinedlimits are used as symptoms, indicating poor operation/performance ofthe flow cytometer. In order to evaluate the differences a Signal Meanis calculated (Gauss mean).

Library

The information supplied by the manufacturer includes a “library” forfault finding. In the library a number of symptoms are related toadequate advices indicating to the user the actions to be taken in orderto remedy any poor operation/performance. In a preferred embodiment thelibrary is stored on a disk, preferably a CD-rom or similarhigh-capacity disk. An example of the stored information is shown onpages 11-14 as table No. 1. The example according to table 1 discloses apresently preferred way of displaying the symptoms and diagnosticrecommendations. 14 different situations or so-called “versions” aredescribed in the table. No. 0.0, i.e. the first one, is the perfectcase, all values are within limits and close to the optimum. All of thenext 13 cases represent indications for some kind of maladjustment. Thefirst column to the left shows the observed symptoms. (In some cases thesame symptoms appear in different versions a, b, c). The right columnincludes the messages provided to the user in the right side window atthe bottom of FIG. 10. These messages include: a) further detailedsymptoms, b) indications such as possible proposals for diagnosis ofwhat might be the reason for poor performance, and c) what to do toremedy the situation.

Typical symptoms indicating poor performance are: Signal Mean low,Signal Mean too low, Signal Mean much too low, Signal Mean high, SignalMean too high, PHA is wide, PHA is low, PHA skewed or double,

Typical diagnosis may be:

electronic gain too low or too high: needs adjustment of the gain in theelectronic signal processing means;

obstructions or clogging in the flow system;

sheath flow too low, too high, or much too high: adjustment of thesample flow rate needed;

incorrect mixing of sample and dye;

defect lamp;

maladjustment of lamp or microscope; needs optical adjustment oralignment of the lamp and/or the microscope;

Preferred embodiment

The principles of a preferred embodiment of the present invention isshown schematically in FIG. 16. The input to the instrument is astandard fluid sample 161 and an information carrier such as a disk 162,comprising information on the lot and software.

The following actions/operations are carried out:

Every 2 hours a computer prompt tells the user to insert a standardsample 161 (from a lot delivered by the supplier)

the flow cytometer measures the standard sample a plurality of times,e.g. three times, i.e. obtains three counts, c₁, c₂, c₃, each of whichrepresents the number of particles passing by during a predeterminedmeasurement period of a few seconds, e.g. 2 sec, (preferably, a periodaccording to normal operation mode of the instrument). The count isassigned a consecutive record or registration number, e.g. No 58.1,58,2, 58.3, 59.1 . . .

The data processing equipment including appropriate software calculates

the number of particles c₁, c₂, c₃

the mean count, c=(c₁+c₂+c₃)/3, (count in FIG. 5, also shown as 165 inFIG. 16)

the signal size

the mean signal size (Signal Mean in FIG. 6), and

the standard deviation is calculated from the three measurements,

s={square root over (((c ₁ −c)²+(c ₂ −c)²+(c ₃ −c)²)/2)}−or theCoefficient of Variation: 100%*s/c

(CV is shown in FIG. 7, compared with theoretical CV that is calculatedaccording to Poisson sampling statistics); also shown as 167 in FIG. 16;

an accumulated mean value of all counts obtained by the instrument:

Σc ₁, (Count Cumulative in FIG. 8 and 168 in FIG. 16)

The calculated results are compared to the information delivered withthe lot (by the supplier of the lot) including upper and lower optimaloperating limits (reference numbers 51, 52, . . . in FIGS. 5-8) (e.g.mean signal size, count, CV),

and comments to each measurement and mean results are displayed to theuser e.g. as shown in the list of results in FIG. 9, in order to drawthe operator's attention to any poor performance. For the sake ofclarity, extracts from the list of results are redisplayed in table 3.

On request the data processing equipment (with appropriate software) ofthe flow cytometer displays a PHA-diagram on the attached monitor 30indicating the signal size.

Each count (ref. number 50 I FIG. 5) is compared to the Lot count c₀(53), which is a count number supplied with the lot and representing theactual number of particles in the present lot. Accordingly, the presentcount ideally should equal the lot count (53). Lot limits (51, 52)indicating admissible variations are also supplied with the lot. FIG. 5shows the count (50) compared with lot count (53) for a plurality ofstandard samples 0-72. The horizontal axis indicates the registeredsample No. and the vertical axis indicates the count per 1000 particles.The allowable variations are indicated by upper and lower lot limits(51, 52) . In the present case the lot count/1000 is 396 and the limits(51, 52) are set to 371 and 421. The bold line or track (50) indicatesthe actual counts/1000 found. They appear to be within the limits (51,52).

FIG. 6 shows a Signal Mean (60) compared with a lot signal (63) andupper and lower limits (61, 62). In order to illustrate (as an example)a possible failure or defect, the attention is drawn to sample No 48: Asignificant decrease in signal seems to appear after sample No. 48. Whencomparing to the detailed result it appears that somehow the gain wasdecreased from 625 to 615.

FIG. 7 shows as an example a sequence of calculated CV/R (ref. number 70I FIG. 7) compared with the theoretical value: CV/R=1 (72). An upperlimit (71) indicates the admissible variation.

FIG. 8 shows a Count Cumulative (80) compared with theoretical values.The accumulated mean value is compared to curved limits (81, 82) (whichare p% confidence limits) confining a gap getting narrower as the samplenumber increases. However, a batch of samples (so-called “lot”) has alimited number of samples and a new lot may have a different number ofparticles per ml. In order to avoid a displacement of all the predefinedvalues, e.g. the theoretical mean, (the so-called lot count), and thelimits, it is preferred that the accumulated mean value (80) and thelimits (81, 82) are normalised, i.e. divided by the Lot Number, a lotmean count in order to allow for continuous watch, also when the lot ischanged.

EXAMPLE

When the user gets a message or remark, e.g. as indicated in table 1 forthe records 19, 49, 51, 58 and 59, he should as soon as possible studythe latest measurement results. The software is “windows®”-based,providing for display of several “windows”. FIG. 9 shows a list of“check measurements” or results of the same kind shown in table 3. Thehighlighted sample No. 58 has got a remark. Accordingly, the user hasasked for a display of “Result details for 58”. The result details aredisplayed in table form (also shown in table 2 page 15) including sixrows (comprising the three measurements (rep 1, rep2, rep3), an averagevalue, CV% (a theoretical standard deviation), and the lot average), andeight columns (comprising the values found for the count, R, SignalMean, (58), Signal Width (8), Z (a value which quantifies thediscriminating space (separation) between noise and signals),Discriminator, Noise Level, and Remarks). In a third window the lot dataare displayed. Further, the display facilities allow for the display ofa measured PHA-diagram and a theoretical, desired PHA-diagram (FIG. 10).A print-out of the displayed PHA-diagrams is shown in FIG. 11. As itappears from the screen print in FIG. 10, the user may select anyregistered measurement, e.g. No 58.2, which is stored in the datastorage means as a sequence of numbers, comprising the ordinates for thePHA-curve obtained for No 58.2. The PHA-curve is displayed with theoptimal PHA-curve. It is obvious that the signal Mean is too low (cf.FIG. 6). The bottom section of the display comprises three smalladjacent windows, a left window in which the symptom “Signal Mean is toolow” is highlighted, a centre window showing the symptomatic picture ofthe PHA-curves, and a right-side window showing details of symptoms andindications, comprising recommendations for how to remedy the poorperformance. Further examples of symptoms and recommendations are shownin table 1 on pages 11-14. In the below table “QCHECK LIBRARY” is ashort notation for Quality check library.

FIGS. 12-14 show as examples PHA-diagrams for various situations. Thevertical lines 91 and 92 are lower and upper lot limits for PHA for themeasured FMA-sample. The grey PHA curve 93 shows how the PHA ideallyshould look, the dark curve 94 shows the curve of the actually measuredsample.

The PHA-diagram of FIG. 12 illustrates a situation characterised in thatthe Signal Mean is too high. Measured Signal Mean Value is outside highlimit of lot signal Mean Value. PHA is high, narrow and resembles LotPHA. The indications relating to this situation are: Gain setting may betoo high, check gain in result list, changed? Sheath flow may be low.Has sheath flow been reduced? check. If flow is OK: reduce gain.

The PHA-diagram of FIG. 13 illustrates a situation characterised in thatthe Signal Mean is high, PHA is wide. Measured Signal Mean Value may bewithin or outside limit, and higher than lot signal Mean Value. PHA islower and wider than Lot PHA. The indications relating to this situationare:

Sheath flow may be low. Has sheath flow been reduced? check. Reset flowto 10-10.5 ml/min at earliest convenience. Finally set gain correctly(i.e. Measured Signal Mean shall be close to Lot Signal Mean +/−2).

The PHA-diagram of FIG. 14 illustrates a situation characterised in thatthe PHA is skewed or double. Measured Signal Mean Value may be within oroutside limit of lot signal Mean Value. PHA is skewed or double, lowerand wider than Lot PHA. The indications relating to this situationare: 1) Microscope adjustment is not OK 2) Sheath flow may not becorrect Check sheath flow and reset if necessary to 10-10.5 ml/min.Readjust microscope gently for narrow and high PHA and max signal mean.Finally set gain correctly (Measured Signal mean is close to Lot SignalMean +/−2).

It appears from the above examples that the method according to thepresent inventions allows the user himself to perform many ordinaryadjustments of the instrument Thus it is ensured that the instrumentwill operate at perfect performance substantially all the time.

TABLE 1 EXPLANATIONS IN QCHECK LIBRARY PER 15 AUG. 1997 HEADERDESCRIPTIONS Signal Mean Measured Signal Mean Value is within limits andclose to Lot Signal Mean Value. is correct (a) PHA is high, narrow andresembles Lot PHA. Indications: (1) Gain setting is OK (2) Sheath flowis OK. (3) Microscope adjustment is Ok. (4) Lamp is OK. (5) Noobstructions in flow cell. Note: If measured PHA is narrow andpositioned correctly then it is of no significance if measured PHA is alittle lower or higher than Lot PHA (Ver. 0.0) Signal Mean MeasuredSignal Mean Value is within limits but lower than Lot Signal Mean is low(a) Value. PHA is high, narrow and resembles Lot PHA. Indications: (1)Gain setting may be a little low. Check gain in result List. Changed?(2) Sheath flow may be high. Has sheath flow been increased? Check. (3)Lamp alignment may be disturbed. What to do: No urgent adjustments. Ifflow is OK (9.5-10.5 ml/min.): Watch for any further changes thefollowing days. When appropriate: Increase gain to correct setting(Measured Signal Mean is close to Lot Signal Mean, ±2). (Ver. 1.0)Signal mean Measured Signal Mean Value is outside low limit of LotSignal Mean value. PHA is too low is high(er), narrow and resembles LotPHA. Indications: (1) Gain setting may be too low. Check gain in resultList. Changed? (2) Sheath flow may be much too high. Has sheath flowbeen increased? Check. (3) Lamp may have failed. What to do: Findproblem at your earliest convenience. If gain is unchanged and flow isOK (9.5-10.5 ml/min.): Realign lamp for maximum Signal Mean Value orreplace lamp. Finally set gain correctly (Measured Signal Mean is closeto Lot Signal Mean, ±2). (Ver. 2.0) Signal mean Measured Signal MeanValue is within limits but higher than Lot Signal Mean is high (a)Value. PHA is high, narrow and resembles Lot PHA. Indications: (1) Gainsetting may be a little high. Check gain in result List. Changed? (2)Sheath flow may be low. Has sheath flow been reduced? Check. What to do:No urgent adjustments. When appropriate: If flow is OK (9.5-10.5ml/min.) gain is reduced to correct setting, i.e. Measured Signal Meanis close to Lot Signal Mean, ±2. (Ver. 3.0) Signal mean Measured SignalMean Value is outside high limit of Lot Signal Mean Value. is too highPHA is high, narrow and resembles Lot PHA. Indications: (1) Gain settingmay be too high. Check gain in result List. Changed? (2) Sheath flow maybe low. Has sheath flow been reduced? Check. What to do: Find problem atyour earliest convenience. If flow is OK (9.5-10.5 ml/min.) gain isreduced to correct setting, i.e. Measured Signal Mean is close to LotSignal Mean, ±2. (Ver. 4.0) Signal Mean Measured Signal Mean Value iswithin limits and close to Lot Signal Mean Value. is correct (b) PHA ishigh, narrow and resembles Lot PHA. See also: “Signal Mean is correct(a)”. This is an example of measured PHA a little lower than Lot PHA.Variations in PHA-heights - insignificant to milk measurements - may becaused by minor differences in optics, lamp and microscope adjustments.(Ver. 5.0) Signal Mean Measured Signal Mean Value is within limits buthigher than Lot Signal Mean is high (b) Value. PHA is high, narrow andresembles Lot PHA. Indications: (1) Gain setting may be a little high.Check gain in result List. Changed? (2) Sheath flow may be low. Hassheath flow been reduced? Check. (3) Microscope may need adjustment Whatto do: If flow is not OK: Repair problem at your earliest convenience.Increase flow to 9.5-10.5 ml/min. Check or readjust microscope gentlyfor max. Signal Mean. Finally set gain correctly, i.e. Measured SignalMean is close to Lot Signal Mean, ±2. (Ver. 6.0) Signal Mean MeasuredSignal Mean Value may be within or outside limit and higher than Lothigh, Signal Mean Value. PHA is lower and wider than Lot PHA. PHA wideIndications: (1) Sheath flow may be low. Has sheath flow been reduced?Check. What to do: Reset flow to 9.5-10.5 ml/min. at your earliestconvenience. Finally set gain correctly, i.e. Measured Signal Mean isclose to Lot Signal Mean, ±2. (Ver. 7.0) Signal Mean Measured SignalMean Value is within limits but lower than Lot Signal Mean is low (b)Value. PHA is high, narrow and resembles Lot PHA. Indications: (1) Gainsetting may be a little low. Check gain in result List. Changed (2)Sheath flow may be high. Has sheath flow been increased? Check. (3) Lampalignment may be disturbed. (4) Microscope may need adjustment. What todo: If flow is not OK: Repair problem at your earliest convenience.Reduce flow to 9.5-10.5 ml/min. Check or readjust microscope gently formaximum Signal Mean. Finally set gain correctly, i.e. Measured SignalMean is close to Lot Signal Mean, ±2. (Ver. 8.0) Signal Mean MeasuredSignal Mean Value is within limits but lower than Lot Signal Mean is low(c) Value. PHA is high, narrow and resembles Lot PHA. Indications: (1)Gain setting may be a little low. Check gain in result List. Changed?(2) Sheath flow may be high. Has sheath flow been increased? Check. Whatto do: If flow is not OK: Repair problem at your earliest convenience.Reduce flow to 9.5-10.5 ml/min. Finally set gain correctly, i.e.Measured Signal Mean is close to Lot Signal Mean, ±2. (Ver. 9.0) PHAlow, wide Measured Signal Mean Value may be within or outside low limitof Lot Signal Mean Value. PHA is skewed, lower and wider than Lot PHA.Indications: (1) Microscope adjustment is not OK. What to do: Repairproblem at your earliest convenience. Check that gain is OK; do notchange it. Check that flow is OK, 9.5-10.5 ml/min. Readjust microscopegently to obtain narrow and high PHA and a maximum Signal Mean Value.Finally set gain correctly, i.e. Measured Signal Mean is close to LotSignal Mean, ±2. (Ver. 12.0) PHA skewed Measured Signal Mean Value maybe within or outside low limit of Lot Signal or double Mean Value. PHAis skewed/double, lower and wider than Lot PHA. Indications: (1)Microscope adjustment is not OK. (2) Sheath flow may not be correct.What to do: Repair problem at your earliest convenience. Check sheathflow and reset if necessary to 9.5-10.5 ml/min. Readjust microscopegently to obtain narrow and high PHA and a maximum Signal Mean Value.Finally set gain correctly, i.e. Measured Signal Mean is close to LotSignal Mean, ±2. (Ver. 13.0) Signal Mean Measured Signal Mean Value isoutside limit of Lot Signal Mean Value. PHA is is much too low high,narrow and resembles Lot PHA. Indications: (1) Gain setting may be alittle low. Check gain in result List. Changed? (2) Sheath flow may bemuch too high. Has sheath flow been increased? Check. (3) Microscope maybe maladjusted. (4) Lamp alignment or replacement may be needed. What todo: Repair problem at your earliest convenience. Check and reset flow ifnecessary to 9.5-10.5 ml/min. Adjust lamp to maximum DC-level on milksample. Adjust Microscope gently to maximum on DC-level on milk sample.Fine tune microscope very gently to max. Signal Mean Value with FMAsample. Finally set gain correctly, i.e. Measured Signal Mean is closeto Lot Signal Mean, ±2. If gain has to be increased significantly:Replace lamp. (Ver. 14.0)

In a specifically advantageous embodiment of the present invention theregistered data and/or provided parameters may be transferred by a modemand attached communication means to a service centre at a supplier ormanufacturer, who in return may provide recommendations for remedyingthe defect and/or send corrective messages controlling the settings ofthe apparatus. In this way also difficult cases in which the user wouldtend to ask for the visit of a service engineer may be taken care of byremote control.

Compensation for Agglomeration of Particles in the Standard Sample

The PHA diagram further provides for a compensation for agglomeration ofparticles in the standard sample. Experience has shown that occasionallythe standard samples may include some particles which have agglomeratedtwo by two into doublets, or three by three into triplets. The biggerparticles, the bigger signals. Accordingly, the PHA-diagram may look asindicated in FIG. 15.

The first top (101) represents single particles (singlets) in a numberof 1166; the second top (102) represents double particles (doublets) ina number of 429, and the third top (103) represents triplets in a numberof 77. Accordingly the program, i.e. the software controlling theinstrument and evaluating the measurements may be arranged to compensatefor the agglomerated particles by adding the doublets and triplets twiceand three times, respectively.

The formation of doublets and triplets (and possibly other multiple) isbelieved to depend on the age of the particles, the shaking of thesample as well as the temperature of the sample. It is difficult topredict the amount of doublets and triplets in the standard samples andaccordingly the above method of compensation solves a current problem.

It is obvious to people skilled in the art that embodiments according tothe invention and described above may be varied in several ways withinthe scope of protection as defined in the following patent claims.

TABLE 2 Result details for 58 Signal Signal Discrimi- Noise Count R MeanWidth Z nator Level Remarks rep 1 418 2.67 58 8 3.3 29 4 signal low rep2 399 2.74 58 8 3.4 28 4 signal low rep3 399 2.73 58 8 3.7 25 4 signallow Average 405 2.71 58 8 3.4 27 4 signal low CV(%) 271 0.0 Lot 396 2.7573 7144511 Average

Fossomatic Quallty Check - Results Signal Signal No. Date Time Count CVR Mean CV Width Remarks Lot No Gain 1 June 6, 1997 12:36:15 393 0.832.76 73 0.0 8 OK 7144511 622 2 June 6, 1997 12:37:30 402 5.45 2.73 730.0 8 OK 7144511 622 3 June 6, 1997 12:37:51 389 6.20 2.77 73 0.8 8 OK7144511 622 . . . 19 June 6, 1997 17:01:28 374 6.16 2.83 72 0.8 9 aremark 7144511 625 20 June 6, 1997 17:02:21 390 0.70 2.77 73 0.0 8 OK7144511 625 21 July 7, 1997 08:38:22 419 1.19 2.67 72 0.0 8 OK 7144511625 . . . 48 July 10, 1997 14:33:30 400 1.93 2.73 64 0.9 8 OK 7144511625 49 July 10, 1997 16:17:36 394 2.28 2.75 49 1.2 8 signal low 7144511615 50 July 10, 1997 17:34:39 389 6.69 2.77 68 0.0 8 OK 7144511 615 51July 11, 1997 11:02:47 391 1.35 2.76 62 2.5 8 signal low 7144511 615 . .. 57 July 15, 1997 06:51:50 398 2.47 2.74 62 0.9 8 OK 7144511 615 58July 15, 1997 08:52:45 405 2.71 2.71 58 0.0 8 signal low 7144511 615 59July 15, 1997 10:54:27 388 2.69 2.77 58 1.0 8 signal low 71445i1 615

What is claimed is:
 1. A method of checking the performance of a flowcytometer instrument (10), in which flow cytometer instrument the numberof particles or cells in a fluid flow are counted by providing datarepresenting a PHA diagram (Pulse Height Analysis) of registered pulses,the method comprising the steps of: providing a lot of standard samples,having a standard fluid, including only one type of substantiallyuniform microbeads, providing optimal data representing an optimal(desired) PHA diagram (PHA0) of the pulses registered, when measuring astandard sample from said lot on a reference flow cytometer instrument,said optimal data representing an optimal (desired) PHA diagram (PHA0)being stored in one of the means of the group consisting of: a memory inthe flow cytometer instrument to be checked (10) itself, a memory indata processing means connected to the flow cytometer instrument to bechecked (10), means from which the optimal data may be imported into theflow cytometer instrument to be checked (10), and means from which theoptimal data may be imported into a data processing means connected tothe flow cytometer instrument to be checked (10), measuring a standardsample on the flow cytometer instrument (10) to be checked, providingsample data representing a PHA diagram (PHAS) for the pulses registeredduring the measurement of the standard sample on the flow cytometerinstrument (10) to be checked, comparing the sample data to the storedoptimal data, and analysing and/or evaluating said sample data andstored optimal data in order to determine any poor or faulty operationof the flow cytometer instrument to be checked.
 2. The method accordingto claim 1, wherein a standard sample is measured at least three timesin the flow cytometer instrument to be checked, and wherein the steps ofcomparing and analysing comprises the steps of: processing the sampledata representing PHA diagrams (PHAS) so as to calculate and/or providea number of parameters selected from the group consisting of: a particlecount, a plurality of particle counts on the same sample, a mean count,a standard deviation s and/or Coefficient of Variation CV, based onrepeated/consecutive measurements on the same sample and substantiallyat the same time, a signal mean value, a signal width (width of thebell-curve in the PHA-diagram), providing optimal values ofcorresponding parameters for the standard sample of the standard fluidmeasured on the reference flow cytometer instrument, comparing theselected parameters for the actual measurement of the standard sample tothe optimal values of corresponding parameters of the standard fluidwhen measured on the reference flow cytometer instrument, analysing saiddata representing actual parameters and optimal parameters to estimatewhether the flow cytometer instrument to be checked is operatingsubstantially optimally, or is not operating substantially optimally,registering any off-limit deviations from optimal operation.
 3. Themethod according to claim 2, wherein the microbeads are unstained untilthey are applied in the flow cytometer instrument to be checked (10). 4.The method according to claim 2, wherein registered off-limit deviationsare considered as symptoms which are indicated to the user.
 5. Themethod according to claim 4, wherein a symptom indicated to the user isassociated with a proposal for remedying the defect, expected togenerate the symptom.
 6. The method according to claim 2, wherein theprovided data and/or provided parameters are transferred by a modem andcommunication means to a service centre at a supplier or manufacturer,who in return may provide recommendations for remedying the defectand/or send corrective messages controlling the settings of the flowcytometer instrument to be checked (10).
 7. The method according toclaim 1, further comprising the steps of analysing the PHA diagram(PHAS) of the measured standard sample in order to observe anyagglomerated microbeads in the measured standard sample, and if PHAdiagram reveals further maxima for signals sized above the firstbell-formed area comprising a first maximum, adjusting the number ofmicrobeads calculated from the area of the first maximum in the PHAdiagram (PHAS) using calculations of the number of microbeadsrepresented by an adjacent, second area of a second maximum in the PHAdiagram by adding twice the number of microbeads calculated in thesecond area to the number of microbeads calculated in the first area. 8.The method according to claim 7, wherein the number of microbeadscalculated from the areas of the first and second maxima in the PHASdiagram is adjusted using calculations of the number of microbeadsrepresented by an adjacent, third area of a third maximum in the PHASdiagram by adding three times the number calculated in the third area tothe number calculated in the first and second area.
 9. The methodaccording to claim 2, characterised by providing an accumulated meanvalue (80), and curved limits (81, 82) (which are p% confidence limits)confining a gap getting narrower as the sample number increases.
 10. Themethod according to claim 9, wherein the accumulated mean value (80) andthe limits (81, 82) are normalised, divided by the Lot Number (c₀) beinga lot mean count.
 11. The method of claim 1, further comprising the stepof providing a standard kit comprising a standard fluid including aplurality of substantially uniform microbeads and further comprisesassociated data means for accessing data representing information on thecharacteristics of the standard fluid and specifically the content ofmicrobeads.
 12. The method of claim 11, wherein the step of providing astandard kit includes using unstained microbeads.
 13. The method ofclaim 11, further comprising the step of delivering the fluid as a batchof standard samples in a plurality of containers of prescribed sizecomprising a prescribed volume, the fluid comprising water, additivesand a number of plastic beads, and that associated data means includeinformation on the number of plastic beads in the fluid.
 14. The methodof claim 11, further comprising the step of accessing information on thecharacteristics of the standard samples, as well as informationcomprising a library of symptoms indicating poor performance andrecommendations for how to remedy any poor performance, and/or anyprecautions to be taken.